Epigenetic silencing is often altered in cancer and is a target for drug discovery. Unbiased screens in live cells are performed to identify potential novel targets of epigenetic therapy, and these screens have identified drugs that were not previously recognized to be involved in epigenetic reactivation of gene silencing such as cardiac glycosides and a CDK9 inhibitor. Recently, our lab performed a whole genome siRNA screen in combination with DNMT inhibition. One of the top targets revealed in this screen was the splicing factor SF3B1. SF3B1 is a well-known crucial splicing factor and is mutated in several cancers. However, its role in epigenetic regulation has not been well studied. I propose SF3B1 is a novel target for epigenetic therapy in cancer. In the YB5 colon cancer cell line where GFP is under the control of a methylated CMV promoter, I validated the screen results and found 0%, 1.0% and 5.3% GFP+ cells after treatment with siControl, siSF3B1 or the DNA methyltransferase inhibitor decitabine (DAC), respectively. DAC and siSF3B1 were synergistic, inducing 17.2% GFP+ cells. This synergy was also seen in an additional live cell assay and with other SF3B and SF3A family proteins. RNA-Seq analyses showed 423 genes upregulated by siSF3B1, 430 genes induced by DAC, and 1190 induced by the combination. siSF3B1 resulted in aberrant splicing of 695 genes, but there were only 27 genes overlapping between splicing alterations and gene expression changes, suggesting different mechanisms. Genes regulated upon siSF3B1 treatment were enriched for the TATA motif in their promoters, and the TATA-Box binding protein (TBP) was among the genes differentially spliced after siSF3B1. DNA methylation analyses showed demethylation synergy between siSF3B1 and DAC. Finally, the effects of siSF3B1 were phenocopied by treatment with the pan-SF3B inhibitor Pladienolide B (PB). GFP was reactivated in two separate colon cancer cell lines upon treatment with PB with synergistic activation when combined with DAC in YB5 cells. Thousands of genes were regulated and alternatively spliced with PB treatment alone, and among the differentially spliced genes was TBP. Furthermore, PB treatment with DAC induced demethylation significantly more than with DAC treatment alone. Genes regulated upon SF3B1 loss and inhibition were enriched for p53 target genes. Indeed, there was reduced cell proliferation and cell cycle arrest when SF3B1 was inhibited. This study demonstrates that the splicing factor SF3B1 has unexpected effects on gene transcription and targeting SF3B1 is synergistic with DNA methylation inhibition suggesting clinical potential for the combination. / Biomedical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/1069 |
| Date | January 2019 |
| Creators | Deliard, Sandra |
| Contributors | Sapienza, Carmen, Issa, Jean-Pierre, Engel, Nora, Soboloff, Jonathan |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 127 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/1051, Theses and Dissertations |
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